Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The invention relates to a cell-based drug delivery system comprising
magneto nanoparticles, erythrocytes and a fusion-protein, its preparation
and uses thereof in particular as a delivery system for biologically
active compounds.

Claims:

1. A compound delivery system to a target cell comprising: an
haemoglobin-deprived erythrocyte, a magnetic or super-paramagnetic
nano-particle located inside the erythrocyte and a protein located on the
membrane of the erythrocyte able to induce the fusion of the membrane of
said erythrocyte to the membrane of the target cell.

2. The system according to claim 1 wherein the magnetic or
super-paramagnetic nano-particle is of γFe2O.sub.3.

3. The system according to claim 1 wherein the magnetic or super-magnetic
nano-particle has a diameter between 5 to 100 nm.

4. The system according to claim 1 wherein the magnetic or super-magnetic
nano-particle is covered by a surfactant.

5. The system according to claim 1 wherein the magnetic or super-magnetic
nano-particle is further modified to comprise an additional agent.

6. The system according to claim 5 wherein the additional agent is
selected from the group consisting of an amino or carboxy or esterified
group covalently bound to a fluorescent or non-fluorescent functional
group.

7. The system of claim 1 wherein the protein is a virosome-derived
protein.

8. The system according to claim 7 wherein the virosome is from an
influenza virus virosome or a vesicular stomatitis virus virosome or an
Epstein Barr virus virosome.

9. The system according to claim 1 being loaded with a pharmacological or
biological therapeutic compound.

10. The system according to claim 9 wherein the pharmacological or
biological therapeutic compound is selected from the group consisting of:
antiretroviral compound, small molecule, pro-drug, phosphorylated
pro-drug or a chemiotherapeutic compound.

11-12. (canceled)

13. A method for the preparation of the system of claim 1, comprising: a)
depriving isolated erythrocyte of haemoglobin; b) incubating under
appropriated conditions the haemoglobin-deprived erythrocyte with a
protein able to induce the fusion of the membrane of said erythrocyte to
the membrane of a target cell, thus obtaining an erythrocyte-protein
complex; c) purifying said complex; and d) incubating the purified
complex with magnetic or super-paramagnetic nano-particles under
appropriate conditions.

Description:

FIELD OF THE INVENTION

[0001] The invention relates generally to a new cell-based drug delivery
system and more specifically to "erythro-magneto-virosomes" which have
the capacity to encapsulate therapeutic compounds with different
biological, pharmacological and chemical characteristics, such as
anti-retroviral compounds, small molecules, phosphorylated pro-drugs or
chemotherapeutics. Such system is also able to specifically drive the
pro-drug at the target tissues or organs by application of specific
electromagnetic fields at cyclotron resonance of iron ions. The invention
concerns also the method of preparation and uses of the cell-based system
in particular as delivery system.

BACKGROUND ART

[0002] Drug delivery has been greatly improved over the years by means of
chemical and physical agents that increase bioavailability, improve
pharmacokinetic and reduce toxicities. At the same time, cell based
delivery systems have also been developed. The use of cells as
therapeutic carriers has increased in the past few years and has
developed as a distinct concept and delivery method. Cell-based vehicles
are particularly attractive for delivery of bio-therapeutic agents that
are difficult to synthesize, have reduced half-lives, limited tissue
penetrance or are rapidly inactivated upon direct in vivo introduction.
As a matter of facts, the cell-based delivery system possesses a number
of advantages including prolonged delivery times, targeting of drugs to
specialized cell compartments and biocompatibility. The use of a
physiological carrier to deliver therapeutics throughout the body to both
improve their efficacy while minimising inevitable adverse side effects,
is an appealing perspective that can be applied in many clinical
settings.

[0003] The behaviour of erythrocytes, as a delivery system for several
classes of molecules (i.e., proteins, including enzymes and peptides,
therapeutic agents in the form of nucleotide analogues, gluco-corticoid
analogues), has been studied extensively as they possess several
properties which make them unique and useful carriers. Furthermore, the
possibility of using carrier erythrocytes for selective drug targeting to
differentiate macrophages increases the opportunities to treat
intracellular pathogens and to develop new drugs. Finally, the
availability of an apparatus that permits the encapsulation of drugs into
autologous erythrocytes has made this technology available in many
clinical settings and competitive in respect to other drug delivery
systems.

[0004] The erythrocyte-based drug delivery presents the following
advantages in that: [0005] i) it is especially efficient for releasing
drugs in the circulation for long period of time (weeks); [0006] ii)
erythrocytes have a large encapsulating capacity; [0007] iii)
erythrocytes can be easily processed and can accommodate traditional and
biologic drugs.

[0008] These carriers have also been used for delivering antigens and/or
contrasting agents. Carrier erythrocytes have been evaluated in thousands
of drug administration in humans proving safety and efficacy of the
treatments. Erythrocyte-based delivery of new and conventional drugs is
thus experiencing increasing interests in drug delivery and in managing
complex pathologies especially when side effects could become serious
issues (Pierige F et al 2008; Rossi L et al 2005; Roth et al 2008).
Recently, erythrocytes have been used to encapsulate an antibiotic, the
amikacin, and a sustained release from loaded erythrocytes over a 48
hours period was demonstrated, suggesting a potential use of the
erythrocytes as a slow systemic-release system for antibiotics. The
administration to rats of amikacin encapsulated in erythrocytes leads to
significant changes in the pharmacokinetic behaviour of the antibiotic.
Indeed, a greater drug accumulation is observed in the
reticulo-endothelial system (RES) of organs such as liver and spleen.
This shows that loaded erythrocytes are potentially useful for the
delivery of antibiotics in phagocytic cells located in the RES
(Gutierrez-Millan et al. 2007; Gutierrez-Millan et al. 2008).

[0009] However, one important aspect of cell-based delivery yet to be
fully investigated is the process of actual delivery of cell payload, i.e
which is actually transported in vivo. In this regard, the potential
ability of cell carriers to provide site-specific or targeted delivery of
therapeutics has yet to be fully explored. Development of cell targeting
strategies will further advance cell vehicle applications, broaden the
applicability of this delivery approach and potentiate therapeutic
outcomes.

[0010] In recent years, biomedical research indicated that magnetic
nano-particles can be a promising tool for several applications in vitro
and in vivo. In medicine, many approaches were investigated for diagnosis
and therapy, offering a great variety of applications. Super-paramagnetic
and magnetic nano-particles (magnetite-maghemite) are currently used as
contrast agents for magnetic resonance imaging. Magnetic cell separation,
magnetic resonance imaging (MRI), magnetic targeted delivery of
therapeutics or magnetically induced hyperthermia are approaches of
particular clinical relevance. For medical use, especially for in vivo
application it is of great importance that these particles do not have
any toxic effects or incompatibility with the biological organism.
Investigations on applicable particles induced a variability of micro-
and nanostructures with different materials, sizes, and specific surface
chemistry (Alexiou C et al. 2006a). These particles can also be used as
drug carriers for local chemotherapy, called magnetic drug targeting.
Using an external magnetic field, colloidal nano-particles can be
directed to a specific body compartment (i.e. tumor). The
bio-distribution of magnetic nano-particles can be visualized with X-ray
imaging and then confirmed by histological analysis (Alexiou C et al.
2007). Magnetic drug targeting employing nano-particles as carriers is a
promising cancer treatment avoiding side effects of conventional
chemotherapy (Alexiou C et al. 2006b). The biological application of
nano-particles is a rapidly developing area of the nanotechnology field
that raises new possibilities in the diagnosis and treatment of human
cancers. In cancer diagnostics, fluorescent nano-particles can be used
for multiplex simultaneous profiling of tumour biomarkers and for
detection of multiple genes and matrix RNA with fluorescent in-situ
hybridisation. Super-magnetic nano-particles have exciting potentials as
contrast agents for cancer detection in vivo, and for monitoring the
response to treatment. Several chemotherapy agents are available as
nano-particle formulations. Such formulations have at least equivalent
efficacy and fewer toxic effects compared with conventional formulations.
Ultimately, the use of nano-particles should allow a unique and
simultaneous tumour targeting and drug delivery (Yezhelyev M V et al.
2006).

[0011] The present invention relates magnetic nano-particles and/or
erythrocytes that may be transformed, chemically modified and
compositions comprising them. In particular erythro-magneto-virosomes
were designed. Such systems can act as bioactive pro-drug carriers,
containing phosphorylated and or non phosphorylated compounds, which do
not naturally occur in a human or animal organism, said compounds having
different biological and/or pharmacological properties and chemical
characteristics. The delivery system of the invention can be applied in
many clinical settings and are useful, for example, for the treatment of
neoplastic pathologies or pathologies caused by the infection of a human
or animal virus.

[0012] To develop a targeted cell-based drug delivery system, the authors
have produced erythro-magneto-virosomes. Such system is able to
specifically drive a pro-drug at its target tissue or organ, having a
high efficient fusion capability with the targeted cells. In the present
invention, erythrocytes were engineered with both novel targeting
proteins, also called fusion proteins and nano-particles.

[0013] The targeting proteins, in particular from viral origin, improve
the integration of the system with the cytoplasmatic membranes of target
cells. Such proteins are able to induce the fusion of the membrane of the
delivery system to the membrane of the target cell. Furthermore, by
applying physical methods, it is possible to control the distribution and
location of the system thanks to the nano-particles. In the present
invention, a targeting system using chemically modified nano-particles to
carry the pro-drug to the active site was also developed.

[0014] The "erythro-magneto-virosomes", are engineered erythrocytes
comprising paramagnetic or super-paramagnetic nano-particles and
fusion-proteins. In particular, the nano-particles are inside the cell
while the fusion-proteins are located on the cytoplasmic membrane.
Fusion-proteins are preferably virosome fusion-proteins. Pro-drugs can be
inserted into such engineered erythrocytes therefore functioning as drug
delivery system. Pro-drugs, especially in their phosphorylated forms,
remain stable in the engineered erythrocytes until they reach their
target cells. The target cells are able to integrate the phosphorylated
compounds.

[0015] Surprisingly, the erythro-magneto-virosome delivery system of the
invention has a high fusion efficiency with the cytoplasmic membranes of
target cells due to the presence of fusion-proteins on the membrane of
the erythrocyte. In addition, the delivery system of the invention can be
driven to target tissues or organs when a specific electromagnetic field
is applied due to paramagnetic or super-paramagnetic nano-particles
located inside the system. In particular, the nano-particles are of iron.
Moreover, due to their physic characteristics, the delivery systems of
the invention can function as heat source for in situ thermo-ablative
therapy when a specific electromagnetic field is applied. The delivery
system of the invention can be used as bioactive pro-drug carrier due to
its intrinsic cell-mediated homing mechanisms. Erythro-magneto-virosomes
have the capacity to phosphorylate the incorporated drugs, which remain
stable until the pro-drug is driven into target cells. The different
aspects of targeting that can be applied to erythro-magneto-virosomes
vehicles include physical methods for directing vehicles distribution
(applying appropriate magnetic and electromagnetic field at the cyclotron
resonance of ions), intrinsic cell-mediated homing mechanisms (the
ability to phosphorylate the incorporated pro-drugs) and the engineering
flexibility of erythro-magneto-virosomes to respond to any therapeutic
needs.

[0016] Chemically modified magneto nano-particles bound to compounds, are
engineered maghemite nano-particles opportunely modified to include/link
on their surface specific compounds, i.e. drugs, antibodies, small
molecules, DNA or fluorochromes, driven to the site of action through
external application of specific electromagnetic filed. Chemically
modified magneto nano-particles bound to pro-drug are engineered
nano-particles containing an active site that allows them to efficiently
enter the cytoplasmic membrane.

SUMMARY OF THE INVENTION

[0017] The invention relates generally to new cell-based drug delivery
system and more specifically to "erythro-magneto-virosomes" which have
the capacity to encapsulate therapeutic compounds with different
biological and chemical characteristics (such as antiretroviral
compounds, small molecules, phosphorylated pro-drugs, chemiotherapics),
and to specifically drive the pro-drug at the target tissues or organs
though application of specific electromagnetic fields at cyclotron
resonance of iron ions.

[0018] It is therefore an object of the invention a compound delivery
system to a target cell comprising: [0019] an haemoglobin-deprived
erythrocyte, [0020] a magnetic or super-paramagnetic nano-particle
located inside the erythrocyte and [0021] a protein located on the
membrane of the erythrocyte able to induce the fusion of the membrane of
said erythrocyte to the membrane of the target cell.

[0022] Preferably, the magnetic or super-paramagnetic nano-particle is of
γFe2O3. Still preferably, the magnetic or super-magnetic
nano-particle has a diameter between 5 to 100 nm. Yet preferably, the
magnetic or super-magnetic nano-particle is covered by a surfactant. More
preferably, the magnetic or super-magnetic nano-particle is further
modified to comprise an additional agent.

[0023] The additional agent is preferably selected from the group of an
amino or carboxy or esterified group covalently bound to a fluorescent or
no-fluorescent functional group.

[0024] In a particular embodiment, the protein is a virosome-derived
protein. Preferably, the virosome is from an influenza virus virosome or
a vesicular stomatitis virus virosome or an Epstein Barr virus virosome.

[0025] In another particular embodiment, the system of the invention is
loaded with a pharmacological or biological therapeutic compound.

[0026] Preferably, the pharmacological or biological therapeutic compound
is selected from the group of: antiretroviral compound, small molecule,
pro-drug, phosphorylated pro-drug or a chemiotherapeutic compound.

[0027] It is a further object of the invention the system as described
above for medical use. Preferably it is being administered via aerosol,
parenteral or systemic route.

[0028] It is a further object of the invention a method for the
preparation of the system of the invention comprising: [0029] a)
depriving isolated erythrocyte of haemoglobin; [0030] b) incubating under
appropriated conditions the haemoglobin-deprived erythrocyte with a
protein able to induce the fusion of the membrane of said erythrocyte to
the membrane of a target cell, thus obtaining an erythrocyte-protein
complex; [0031] c) purifying said complex; [0032] d) incubating the
purified complex with magnetic or super-paramagnetic nano-particles under
appropriated conditions.

[0033] It is a further object of the invention a pharmaceutical
composition comprising the system of the invention and pharmaceutically
acceptable diluents, carrier and/or excipients.

[0034] The invention will be now illustrated by means of non limiting
examples referring to the following figures.

[0042] FIG. 8a. Propidium iodide cell cycle FACS analysis of untreated and
5-Aza-2-dC-treated HeLa cells after 48-hours--dot plots (first one
without doublets and the second one with only cell cycle) and related
graphics of cell cycle phases are shown. For the analysis, the
Dean-Jett-Fox alogorithm has been used. A) control-1; B) treated with 2.5
μM of 5-Aza-2-dC; C) treated with erythro-magneto-HA-virosomes
containing 5-Aza-2-dC; D) cell treated with supernatant in which
erythro-magneto-HA virosomes containing 5-Aza-2-dC are resuspended
(control-2).

[0043] FIG. 8b. Propidium iodide cell cycle FACS analysis of untreated and
treated HeLa cells after 96-hours--dot plots (first one without doublets
and the second one with only cell cycle) and related graphics of cell
cycle phases are shown. For the analysis, the Dean-Jett-Fox alogorithm
has been used. A) control; B) treated with 2.5 μM of 5-Aza-2-dC; C)
cells treated with erythro-magneto-HA-virosomes containing 5-Aza-2-dC.

[0048] Influenza virus obtained from the allantoic liquid of 10-days-old
embryonated eggs is pelleted by ultracentrifugation and the pellet is
resuspended in octa(ethyleneglycol)-n-dodecyl monoether (C12E8)
and left overnight to allow for complete solubilisation of the viral
membrane. The suspension is carefully homogenised and ultracentrifugated
to pellet down the viral nucleocapsids. Next, the virosome suspension
(supernatant containing the HA protein) is purified by
ultracentrifugation on a discontinuous sucrose-gradient (50% and 10% of
sucrose). The virosomes concentrate at the interface of the sucrose
layers. After removal of this layer, the virosomes can be dialysed
against buffer and sterilized by filtration.

[0052] The mixture was incubated for 30 min at 37° C. The reaction
was stopped by addition of 10 μl of soybean trypsin inhibitor (10
mg/ml), 2 μl of benzamidine (62 mg/ml), and 1 μl of aprotinin (1
mg/ml) in PIPES buffer. The virus was then pelleted by centrifugation
(55,000×g for 30 min) and resuspended in PIPES buffer. HA0 was
purified from trypsin-treated virus and non-treated virus, respectively,
by solubilisation with Triton X-100 and sucrose density gradient
centrifugation, as described previously. Neuraminidase was not removed by
this procedure but remained at a reduced level.

[0053] The isolated HA (1 mg in 1 ml of PIPES buffer) was resolubilized by
addition of 40 μl of 20% (wt/wt) Triton X-100 and by incubation for 1
h at room temperature.

[0054] Triton X-100 treatment at 4° C. is often used for studies of
lipid rafts to obtain detergent-insoluble glycolipid-enriched complexes.
The present procedure at room temperature (25° C.) was distinct
from that at 4° C. in that HA trimers were not aggregated after
Triton X-100 treatment as examined by gel-filtration chromatography
(Sephacryl S-300, Amersham Biosciences, Uppsala, Sweden; data not shown).

[0055] Reconstitution of HA Vesicles and Kinetic of Fusion Analysis

[0056] A lipid mixture mixed with octadecyl rhodamin (R18) (5:1.0, by
weight) in chloroform/methanol (2:1) was dried and kept under vacuum
overnight. The lipid film was suspended in 0.31 ml of PIPES buffer and
mixed with 0.19 ml of 20% Triton X-100. HA fusion proteins were added to
the lipid solution at 1.4 mg of HA per 1 mg of lipid. To remove the
detergent, the mixtures were dialyzed through Spectrapor membrane tubing
2 (25 mm, Spectrum Laboratories, Rancho Dominguez, Calif.) against 2.0
liters of PIPES buffer containing CaCl2 and MgCl2 at 2 mM each and 3 g of
Bio-beads SM-2 (Bio-Rad) at room temperature for 4 h and then at
4° C. for 80 h. The HA-reconstituted vesicles were purified by
sucrose density gradient centrifugation.

[0057] Total lipid in HA vesicles was quantified by the amount of
octadecylrhodamin (R18) in the vesicles, based on the fact that R18
accounts for 15% of the total weight of lipids in the vesicles. The
vesicles were solubilized by adding 0.1% (final concentration) Triton
X-100 in PBS (phosphate buffer saline), and the fluorescence of R18
(excitation at 560 nm and emission at 590 nm) was measured using an
aliquot of the solution with a spectrofluorometer (RF5300-PC, Shimadzu,
Kyoto, Japan), calibrated with R18 standard solutions containing 0.1%
Triton X-100. The degree of R18 self-quenching in each HA vesicle was
examined by comparison of R18 fluorescence before and after
solubilization of the vesicle with 0.1% Triton X-100 in PBS.

[0059] For the determination of the critical micelle concentration (CMC)
of octal glucoside (OG) different saline buffers ([HEPES]=10 mM and
0<[NaCl]<1 M, pH 7.4) containing 1 μM ANS (aniline naphthalene
sulphonyl chloride) were prepared. The ANS fluorescence (Aex=380 nm,
Aem=490 nm) was recorded on a spectrofluorimeter SPEX (FILIII) connected
to a computer. The buffer containing the ANS was poured directly into a
quartz cuvette, which was placed in the spectrophotometer, maintained
under gentle and continuous stirring, and thermo-stated at 25° C.
(±0.2° C.). The evolution of ANS fluorescence was continuously
monitored during the addition of a concentrated OG solution (200 mM). The
rate of OG addition was controlled by a syringe pump (Perfusor VI;
Braun). The OG concentration in the cuvette was then calculated over the
time using the following equation (equation 1):

[OG]tot=([OG]s*rs*t)/(v.(vo)+(rs*t))(mM) Eq 1:

[0060] in which [OG]s is the OG concentration in the syringe in mM,
[OG]tot is the OG concentration raised in the cuvette at any time in the
experiment, rs is the injection rate of the syringe pump in ml/min, t is
the time in min, and VO is the initial volume in the cuvette in ml.

[0061] Solubilization of VSV

[0062] The solubilization of intact viruses has been followed during the
continuous addition of OG by measuring optical density (OD) at 550 nm on
a Perkin-Elmer (Lambda 2) double-beam spectrophotometer. A volume of 1.4
ml of initial VSV solution was placed in a 1-cm optical quartz cell
thermostated at 25° C. (±0.2° C.) and equipped with a
paddle stiffer. A concentrated OG solution made in the same buffer as the
one used to dilute VSV (i.e., 1 M NaCl, 10 mM HEPES, 1 mM EDTA (ethylene
diammino tetraacetic acid), pH 7.4) was progressively added through thin
tubing connected to a glass precision syringe pump (Perfusor VI; Braun).
The OD was recorded as a function of time; this was correlated to the OG
and VSV concentrations in the cuvette by Eqs. 1 (see above) and 2,
respectively:

[VSV[V]stv]0to,t=1+([-O_G[tVoStV/(0O(GM]s-[OG]tot)2 Eq 2:

[0063] in which [VSV]0 is the initial VSV concentration in the cuvette,
which can be expressed in mg total protein/ml; [VSV],0, is the VSV
concentration in the cuvette at any time in the experiment.

[0064] Removal of the Nucleocapsid

[0065] The solubilized VSV was centrifuged for 1 h at 64,000×g to
spin down the nucleocapsid. The supenatant containing the solubilized
proteins and lipids was collected.

[0066] Reconstitution of VSV Envelope by Dilution

[0067] The reconstitution profiles were obtained by following the
turbidity (OD at 550 nm) at 25° C. (+0.2° C.) during the
continuous addition of free OG buffer to a cuvette containing 1 ml of the
solubilized envelope. A volume of 2.5 ml of buffer was added to the
cuvette over 3 h, giving a final dilution factor of 3.5. The OG and VSV
concentrations were calculated from the time using Eqs. 3 and 4:

=[OG]t.t=[OG]o*(vo)/(vo+(r,*t))(mM) Eq. 3

=LrVSVIItot--[V(SvVo]=+(+(rs*tt)(mg*ovfop)rotein/ml) Eq. 4

[0068] in which [OG]o and [VSV]O are the initial OG and VSV concentrations
in the cuvette in mM and mg/ml, respectively; [OG]tot and [VSV],0, are
the OG and VSV concentrations raised in the cuvette at any time in the
experiment; rs is the injection rate of the buffer in ml/min; t is the
time in min; and vo is the initial volume in the cuvette in ml.

[0069] Reconstitution of VSV Envelope by Absorption of OG on BB SM2

[0070] For functional reconstitution, Bio Beads SM2 were used to eliminate
OG. Ten vials were prepared, each containing 20 mg wet Bio Beads SM2 for
each millilitre of sample. The sample was added to the first vial and
kept under gentle magnetic stirring for 12 min at room temperature
(25° C.). Thereafter, the sample was taken from the vial and
poured into the following vial. The total duration of the elimination is
about 2 h. When needed, R18 was added to the solubilized sample at a
probe-to-lipid ratio of 1% before detergent removal by BB SM2 (see FIG. 1
for Ultra-micrograph of influenza virus and gel electrophoresis of HA
purification from influenza virus).

[0071] Epstein Barr Virus (EBV) and EBV Virosomes Preparation

[0072] Virus-producing P3HR1-C113 cells (ATCC, HTB62) were induced with 30
ng of 12-O-- tetradecanoylphorbol-13-acetate per ml, and after 7 days
virus was collected from the spent culture medium. The medium was
centrifuged at 4,000×g for 10 min to remove cells, 100 μg of
bacitracin per ml was added to the clarified supernatant, and the virus
was pelleted by centrifugation at 20,000×g for 90 min. Pellets were
suspended in 1/250 of the original volume of medium containing 100 μg
of bacitracin per ml, reclarified by centrifugation three or four times
at 400×g, and filtered through a 1.2-μm-pore-size filter
(Acrodisc; Gelman Sciences, Inc., Ann Arbor, Mich.) and stored at -700 C.

[0075] Virosomes were made as following described. Briefly, pelleted
virions were extracted with lysing buffer containing 1% Triton X-114, 10
mM Tris, pH 7.4, 150 mM NaCl, 3 mM NaN3, 1 mM
phenylmethylsulfonylfluoride (PMSF), and 100 U7 ml of aprotinin,
sonicated for 1 min, and then centrifuged at 100,000×g for 1 h. The
supernatant was assayed for protein concentration by using BCA protein
assay reagents (Pierce) and bovine serum albumin as a standard. Extracted
viral protein at a 1:5 (wt/wt) protein:lipid ratio was added to a dried
mixture of L-a-lecithin (egg) and cholesterol (Avanti Inc., Pelham, Ala.)
(molar ratio, 1.7:1). The detergent:lipid molar ratio was adjusted to a
minimum of 6:1, and virosomes were formed by extensive dialysis against
buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 3 mM NaN3, and 1 mM PMSF)
containing Amberlite XAD-2 (Sigma Chemical Co., St. Louis, Mo.) to remove
detergent to below its critical miceilar concentration; the final
dialysis was done against buffer without PMSF or NaN3. The dialysate was
frozen and thawed three times and sieved over Sepharose 2B-300 (Sigma).
Virosomes were collected in the void volume, pelleted by centrifugation
at 16,000×g for 30 min, suspended in buffer, and stored at
-70° C.

[0076] EBV Glycoprotein gp 85 and 350/220 Purification

[0077] DEAE Chromatography.

[0078] DEAE Sephacel (Pharmacia Fine Chemicals, Sweden) was washed with
DEAE buffer (10 mM Tris, 0.1 mM EDTAL3 0.5% Triton, pH 7.8) and then
poured into a Pharmacia 26/40 column to produce a bed volume of
approximately 100 ml. Approximately 130 mg of the soluble extract
prepared from TPA-activated 8-95-8 cells were then poured onto the column
and then eluted with a 0 to 0.5 M linear NaCl gradient in DEAE buffer.
Six-milliliter fractions were collected, and every fifth sample was read
for conductivity with a Beckman conductivity bridge, model RC16B2
(Irvine. CA). All fractions with conductivities between 10,000 and 13,500
ohms were then pooled. Protein determinations on every other tube from
the DEAE column were determined with Coomassie Blue technique, with
bovine serum albumin as the standard protein.

[0079] Ricin Chromatography.

[0080] A 5-ml column was formed with Ricinus communis agglutinin II
(Ricin) bound to agarose beads (E-Y Laboratories, San Mateo. CA). The
pooled extract from DEAE column was then passed through the column, and
the column was washed with 30 ml of PBS plus 0.5% Triton. Glycoproteins
were then eluted from the column with 0.05 M lactose and 0.05 M galactose
in PBS plus 0.5% Triton. Two-milliliter fractions were collected, and
each fraction analyzed for protein as described above. Positive fractions
were then pooled and analyzed for the presence of gp 85 and gp350/220
(see FIG. 2 for gel electrophoresis of EBV viral spike glycoproteind
purification).

[0087] The purified erythro-virosomes can be also lysed by rapid dilution
in 300 ml of ice-cold lysis buffer (5 mM EDTA, 5 mM HEPES, pH 7.4). The
lysed red blood cells (RBC) are then pelleted by centrifugation at 27
000×g for 40 min at 4° C. The pellet is re-suspended in
lysis buffer and pelleted again three times or until the RBC membrane
preparation became white. The resulting lysed RBC virosomes
(haemoglobin-deprived RBCs) are resealed in the presence of both the
magnetic or super para magnetic nano-particles (magnetite-maghemite) and
the appropriate therapeutic doses of compounds (i.e chemical drugs,
antibodies, DNA, small molecules, fluorochromes) at 37° C. for 1
hour.

[0088] The erythro-magneto virosomes are then isolated again by
centrifugation at 27 000×g, suspended in buffer and stored in small
aliquots at -20° C. until required.

[0089] Total lipids in erythro-magneto-virosomes are quantified by the
amount of octadecyl rhodamine (R18) in the haemoglobin-deprived RBCs,
based on the fact that R18 accounts for 15% of the total weight of lipids
in the haemoglobin-deprived RBCs. The erythro-virosomes are solubilized
by adding 0.1% (final concentration) Triton X-100 in PBS, and the
fluorescence of R18 (excitation at 560 nm and emission at 590 nm) is
measured using an aliquot of the solution with a spectrofluorometer,
calibrated with R18 standard solutions containing 0.1% Triton X-100. The
degree of R18 self-quenching in each erythro-virosomes is examined by
comparison of R18 fluorescence before and after solubilization of the
erythro-virosomes with 0.1% Triton X-100 in PBS.

[0090] Method 2

[0091] Engineering Erythrocytes with Fusion Proteins

[0092] Human RBCs are obtained from blood of healthy donors by
centrifugation at 1700 rpm for 10 min at 4° C. Fusion protein (HA
or VSV or EBV viral spike glycoprotein) is added to the solution where
the haemoglobin-deprived RBCs are re-suspended at concentration spanning
from 25 μg to 1.4 mg of viral spike glycoprotein per 1 mg of lipids.
To remove the detergents, the mixtures are dialyzed through Spectrapor
membrane tubing 2 (25 mm, Spectrum Laboratories, Rancho Dominguez,
Calif.) against 2.0 liters of PIPES buffer containing CaCl2 and MgCl2 at
2 mM each and 3 g of Bio-beads SM-2 (Bio-Rad) at room temperature for 4 h
and then at 4° C. for 80 h. The virosomes-reconstituted vesicles
are purified by sucrose density gradient centrifugation.

[0096] The erythro-magneto-virosomes containing the desired therapeutic
compound are driven to the desired specific target site for instance by
applying an external static magnetic field. The fusion of
erythro-magneto-virosomes with target cells may be triggered by an
appropriate external resonating electromagnetic field generated by a
specific apparatus for culturing eukaryotic cells as for instance the one
described in the patent application WO/2007/004073.

[0097] Magneto-Nano Particles

[0098] Binding of Magneto Nano Particles with Therapeutic Compounds

[0099] Paramagnetic or super-paramagnetic nano-particles can be chemically
modified with active group enabling them to be efficiently transported
trough the plasma membrane, thus, carrying the pro-drug in the active
form. Magnetic nano-particles of iron maghemite (γFe2O3)
may be used. The particles have a diameter spanning from 5 to 100 nm (5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nm) and may be covered with
surfactants such as Pluronic, dichlorobenzene, oleic acid,
triphenylphosphine, oleyl amine, trioctylphosphine oxide, KorantinSH,
dodecyltrimethylammonium bromide, didodecyldimethylammonium bromide to
inhibit their aggregation. These iron maghemite nanoparticles can be
further opportunely modified to include/link on their surface specific
active groups such as amino or carboxy groups covalently bound to
fluorescent functional group and/or other compounds. The active groups in
their acetyl, esterified, methylated or acethyl-methyl-esterified forms
can bound any compounds. When nanoparticles are conjugated with target
agents or undergo any surface modification, particle agglomeration as a
result of their large surface-to-volume ratio becomes a primary concern
to avoided. When nanoparticles agglomerate, they not only lose their
intended functionality, but can also be quickly cleared by macrophages or
accumulated in the reticule-endothelial system before they can reach the
target cells. One approach to solving this problem is to modify the
particle surface with poly(ethylene glycol) (PEG) self-assembled
monolayers. Surfaces covered with PEG have proven to be non-immunogenic,
non-antigenic, and protein resistant. While the PEG moiety provides an
efficient system to increase particle circulation time in blood
protecting them from aggregation and from fast clearance, the
nano-particle systems may also be coupled with tumor targeting agents to
be useful for the intended applications. Thus, the PEG moiety immobilized
on nano-particles may also provide an active functional group capable of
conjugating with targeting agents. PEG is used widely to functionalize
proteins and peptides for drug delivery. Research in cell targeting has
also utilized functional PEG molecules conjugated with folic acid on
liposomes. Monofunctional PEG molecules coupled to proteins are known to
prolong the particle circulation time in blood, and reduce
immunogenicity. While functionalized carboxyl or amine PEGs are widely
available, they remain expensive and require chemical modification to
convert to their corresponding silanes. Further, these functional PEGs
are available mainly in high molecular weights, which may inhibit PEG
monolayer self-assembly on the nanoparticle surface due to the labile
nature of PEG molecules. Currently available PEGs can conjugate with only
one type of functional group present in targeting agents, typically
either amine or carboxyl groups, and, in most of cases, they are not
suitable for nanoscaled devices such as nanoparticle systems due to their
high molecule weight. PEG silylation normally occur in organic solution,
whereas conjugation with tumor targeting agents such as folic acid or
antibodies needs to be conducted in aqueous solution. Thus, PEG
self-assembled mono layers (SAM) must be flexible enough to prevent
agglomeration during solvent exchange and remain active in solvents to
provide a terminus for conjugation. Despite the advances in the use of
nanoparticles as contrast agents and drug carriers noted above, a need
exists for nanoparticles that can be surface-modified to function as both
contrast enhancement agents and drug carriers simultaneously, allowing
real-time monitoring of tumor response to drug treatment. The present
invention seeks to fulfil this need and provides further related
advantages. Synthesis of magnetic materials on the nanoscale is a topic
of great currently, because of interest the novel microscopic properties
shown by particles of quantum dimensions located in the transition region
between atoms and bulk solids. Nanoparticles have physical and chemical
properties that are characteristic of neither the atom nor the bulk
counterparts. Quantum size effects and the large surface area of magnetic
nanoparticles dramatically change some of the magnetic properties and
exhibit superparamagnetic phenomena and quantum tunnelling of
magnetization, because below a critical size, nanoparticles become single
domain. Because of the unique physical properties, superparamagnetic
nanoparticles have great potential for several applications in different
areas such as ferrofluids, color imaging, information storage, and cell
sorting. As is known, below a critical size, nanoparticles will exhibit
superparamagnetism. The hysteresis loop is usually used to study the
superparamagnetism of the particles. It is generally considered that once
the hysteresis loop exhibits a curve, we can say that the particle has
superparamagnetism.

[0100] Synthesis of Nanoparticles

[0101] Ferric chloride hexahydrate (FeCl3+6H2O>99%) and
ferrous chloride tetrahydrate (FeCl2+4H20>99%) are used as
iron sources, and aqueous ammonia is used as the precipitator. Distilled
water is used as the solvent. The solution of FeCl3 and FeCl2
is mixed with certain molar ratio. The corresponding phase NH4OH is
slowly injected into the mixture of FeCl3 and FeCl2 under
vigorous stirring. After precipitation, the Fe3O4 particles
must be repeatedly washed and filtered before drying at room temperature
in air atmosphere to form powders. The chemical reaction of
Fe3O4 precipitation can be described as follows:

Fe2++2Fe3++8OH--------Fe3O4+4H2O

[0102] Before the reaction, N2 gas must flown through the reaction
medium to prevent possible oxidation. The reaction is operated in a
closed system to provide a non oxidation environment. Otherwise,
Fe3O4 might also be oxidized as follows:

Fe3O4+0.25O2+4.5H2O-------3Fe(OH)3

[0103] which means the suspension will turn from black to yellow and will
affect the purity of the final production. The Fe3O4
nano-particles prepared by co-precipitation dried at room temperature in
air atmosphere to form a powder, which we called the powder sample.
Fe3O4 powder, 0.0122 g, is dispersed into 6 ml distilled water
to form the aqueous solution and then HCl added to the aqueous solution
to a pH of 2. After ultrasonic shocking of the aqueous solution, the
Fe3O4 nano-particles are dispersed in the distilled water. The
structural properties of Fe3O4 nano-particle powders then are
analyzed by X-ray diffraction (XRD; D/MAX3B) using Cu Ka-radiation. The
XRD intensity data are collected over the range 20°, 2v,
60° at room temperature. The mean size and morphologies of
Fe3O4 particles observed by transmission electron microscopy.

[0104] Magnetic Nanoparticles Coating and Functionalization

[0105] Coating

[0106] Isolated magnetic nanoparticles are than diluted in water at 5-30
mg/ml concentrations. A 5-40% aqueous solution of PEGylated ligand added
to the suspension slowly during continuous sonication at 50-60° C.
until all or most of the monolayer-coated particles form a stable aqueous
suspension when placed a top a handheld magnet. Than the PEG coated
nanoparticles are acidic functionalized (---COOH) by proper reaction
methods with tri-ethylamine (Et3N) and dimethylaminopyridine (DMAP)
as catalyst in CH3CN.

[0107] Functionalization of Magneto-Nanoparticles with Esters Groups

[0108] Acetoxymethyl moieties are selectively linked to the nano-particles
surface through carboxylic functions. Esters are obtained using
acetoxymethylchloride (prepared using standard methods) with
tri-ethylamine (Et3N) and dimethylaminopyridine (DMAP) as catalyst
in CH3CN.

[0110] Drugs, pro-drug or any therapeutic compounds covalently linked onto
the surface of nano-particles by esterification of one of the available
hydroxyl functions of the furanosyl moiety of the drug on the carboxylic
function on the particle surface. If necessary, a short alkyl- or
alkoxyl-spacer may be introduced between the drug and the particle. The
reaction is carried out using suitable carbodiimide activation. To ensure
cell permeability of the drug-nanoparticle conjugate, acetoxymethyl
esters are also introduced, either directly on the nanoparticle surface
(according to the method developed and described before), or trough
further functionalization of the drug molecule.

[0111] The second hydroxyl function of the sugar is used to introduce an
acetoxymethyl group. This double functionalization is obtained by
introduction of suitable semi-permanent protecting groups on the drug
molecule.

[0112] Administration of Magneto-Drugs and Erythro-Magneto-Virosomes

[0113] The administration route of both magneto-drugs (maghemite
nano-particles opportunely modified to include/link on their surface
specific compounds, i.e. drugs, antibodies, small molecules, DNA or
fluorocromes, driven to the site of action through external application
of specific elelctromagnetic filed) or erythro-magneto particles
(erythrocytes containing inside or on their surface maghemite
nano-particles which can be realised at the target tissue or cells and
induce local thermal effect if specific electromagnetic filed is applied)
or erythro-magneto-virosomes. Erythro-magneto-virosomes are magneto
erythrosomes containing on their surface viral fusion proteins from
influenza or para influenza virus essential for increasing the efficiency
of fusion of erythro-magneto-virosomes with cell target cytoplasmatic
membranes to release the therapeutic compounds and/or maghemite,
encapsulated inside the erythro-magneto-virosomes, into target cells for
instance via aerosol, by parental or systemic injection.

[0114] Magneto-drugs, erythro-magneto-virosomes or erythro-magneto
particles can be directed to the site of action through external
application, via laparoscopy micro magneto plaques or by using the
specific apparatus for culturing eukaryotic cells able to produce
specific magnetic and electromagnetic filed at the cyclotron resonance of
ions as described for instance in the patent application WO/2007/004073.

[0115] Method for Driving Drug Delivery System and Generating Hyperthermia
in a Field Free Region Using Superparamagnetic Nanoparticles

[0116] Nano-particles may be magnetically driven to site of action, then
drug may be released by applying appropriate sinusoidal and static
magnetic tuned at iron cyclotron energy resonance. After drug being
released it is also possible to proceed with thermal ablation. Heat
therapies such as hyperthermia and thermoablation are very promising
approaches in the treatment of cancer. Compared with available
hyperthermia modalities, magnetic fluid hyperthermia yields better
results in uniform heating of the deeply situated tumors. In this
approach, fluid consisting of super-paramagnetic particles (magnetic
fluid) is delivered to the tumor. An alternating (AC, alternate current)
magnetic field is then used to heat the particles and the corresponding
tumor, thereby ablating it. However, one of the most serious shortcomings
of this technique is the unwanted heating of the healthy tissues. In our
system we demonstrated that by depositing appropriate static and
alternating magnetic fields, both tuned in order to achieve a particular
iron ion cyclotron frequency, focused heating of the magnetic particles
can be achieved.

[0117] Currently, various types of heat treatment modalities are available
for the treatment of cancer such as microwave, ultrasound, focused
ultrasound, radio frequency (RF) capacitance hyperthermia, RF probe
hyperthermia, and magnetic fluid hyperthermia (MFH). Each treatment
technique has certain limitations. For instance, microwave hyperthermia
has a poor depth of penetration, which makes it unsuitable for treatment
of deep-seated tumors. Compared with microwave, ultrasound has better
penetration depth and focusing abilities, but particular drawbacks of
ultrasound include high energy absorption of the bone and
liquid-containing organs RF capacitance hyperthermia, the major limiting
factor is the inability of the electric field to focus on the tumor, so
that all the tissues that the electric field penetrated are heated. RF
probe hyperthermia has the disadvantage of poor accessibility to the
deep-seated tumors, with a limited accuracy of localization. Finally,
this technique is not suitable for the treatment of large tumors. The
patent application WO/2007/004073 concerns a system that focuses the heat
into very small regions so that focused heating of magnetic particles,
and therefore tumors, can be achieved by limiting the damage to the
collateral healthy tissue. This technique envisages increasing the
permeability of a given ion through a membrane, which is subject to the
20 earth's static magnetic field or to any other static magnetic field
which is arbitrarily chosen, superimposing on this static magnetic field
a variable magnetic field modulated with a frequency F proportional to
the ratio Q/m of charge Q to mass m of the ion in accordance with 25 the
relation which defines the cyclotron frequency Fe, 2rr Fc=Q/m Eo where Q
and m are the charge and the mass of the ion expressed respectively in
Coulombs and in kg, and Eo is 5 the intensity of the static magnetic
field expressed in Tesla. This technique, which envisages the use of
Helmholtz coils for the generation of the variable magnetic field, is
claimed to allow regulation, at a speed and with a degree of precision
hitherto unknown, of 10 the ion exchange between the outside and the
inside of a cellular body. It is well known that, under alternating
magnetic fields, super-paramagnetic (single domain) nanoparticles are
heated as a result of the rotation of the particle itself (Brownian
relaxation) and the rotation of the magnetic moment inside the particle
(Neel relaxation). If a static magnetic field with equal or larger
amplitude is superimposed on the alternating field, the single domain
particles and their magnetic moments will align with the static field. In
this way, Neel and Brownian relaxations will be blocked and the heating
of the particles will be diminished. Has been shown that a static
magnetic field, perpendicular to the AC field, applied to magnetic fluids
(colloidal dispersions of super-paramagnetic iron oxide particles) and it
was observed that heating was significantly reduced when the amplitude of
the static field approached that of the alternating field. These studies
demonstrate that static magnetic fields can be used to modulate the
heating effect of AC magnetic fields on the magnetic domains. In
traditional MFH systems, magnetic particles that have been dispersed into
the tissue are heated by application of AC magnetic fields. In this
process, unselective heating of the particles occurs because all the
particles exposed to the alternating field are heated equally. By
depositing appropriate dc magnetic field gradients on the AC magnetic
fields, one can generate AC field dominant regions and achieve focused
heating of the magnetic particles in these regions. The static field
vectors generated by the solenoids cancel each other at the center of the
system and a region with a very small de magnetic field is formed around
the center, which can be named as the field free region (FFR). If an
alternating magnetic field calculated matching the iron cyclotron energy
resonance in earth static magnetic field shielded condition, is applied
to the space between the solenoids, the alternating field will be
dominant in the FFR and only the magnetic particles inside the FFR will
be heated. The particles outside this region cannot be heated as a result
of the dominance of the static field on the alternating field. The
field-free region explained above can be reduced further (i.e., more
intense heat focus can be obtained) by increasing the current magnitudes
flowing through the dc solenoids (see FIG. 3 for a schematic
representation of drug delivery system driving device.

[0119] Erythro-magneto-HA virosomes were prepared as described above and
were labelled by adding Octadecyl Rhodamine (R18). The Erythro-magneto-HA
virosomes/R18 loaded were added to Hela cells. Total lipid in
erythro-magneto-HA virosomes/R18 was quantified by the amount of
Octadecyl Rhodamine (R18) in their membrane, based on the fact that R18
accounts for 15% of the total weight of lipids on it. The
erythro-HA-virosomes/R18 were solubilized by adding 0.1% (final
concentration) Triton X-100 in PBS, and the fluorescence of R18
(excitation at 560 nm and emission at 590 nm) was measured using an
aliquot of the solution with a spectrofluorometer (RF5300-PC, Shimadzu,
Kyoto, Japan), calibrated with R18 standard solutions containing 0.1%
Triton X-100. The degree of R18 self-quenching in each erythro-HA
virosomes was examined by comparison of R18 fluorescence before and after
solubilization of the erythro-HA virosomes with 0.1% Triton X-100 in PBS.
Erythro-magneto-HA virosomes were added to Hela cells. The kinetic of
erythro-magneto-HA virosomes fusion with Hela cells was calculated as %
of R18 fluorescence dequenching (FDQ) using 560 and 590 nm as excitation
and emission wavelengths respectively (see FIG. 4).

[0124] 1×106 erythro-magneto-HA virosomes were incubated with
lysis buffer (10 mM Tris, 0.1 mM EDTA, 1 mM MgCl2) to release the
incorporated 5-Aza-2-dC drug and used for HPLC analysis or stored in a
-80° C. freezer until analysis. Stock standard solution of
decitabine at concentration of 5, 10 and 25 μM were prepared
individually in methanol and stored at -80° C. conditions until
analysis.

[0125] For preparation of calibration curves concentrations of 1, 5, 10
and 15 mμM were used for decitabine. A mixed internal standard
solution in water was freshly prepared on the day of analysis at 5 μM
for decitabine. The peak area ratios of sample/5-Aza-2-dC (decitabine,
standard) was used for quantitation. The limit of detection (LOD) was
defined as three times the signal-to-noise ratio. The lowest limit of
quantitation (LLOQ) was defined as the lowest level of analyte that could
be reliably detected and reproducible with a precision of ≦20% and
accuracy of 80-120% (see FIG. 5).

[0126] Instrumentation

[0127] The HPLC system comprised of a Dionex (Sunnyvale, Calif., USA) 3000
Ultimate series LC connected to a linear ion Trap LTQ-Orbitrap (Thermo
Fisher Scientific, USA) mass spectrometer, equipped with an electrospray
ion source. Data were acquired and processed with Excalibur 2.1 software.
Compounds were separated on a Gemini C18 (150 mm-2.0 mm I.D.) and 3 μm
particle size (Phenomenex, Torrance, Calif., USA) protected by a
Phenomenex Gemini C18 (4.0 mm-2.0 mm I.D.) and 3 μm particle size
guard cartridge. The HPLC method used gradient elution; mobile phase
solvent A was water with 0.1% formic acid and mobile phase B was
acetonitrile with 0.1% formic acid. The initial mobile phase composition
of 92% solvent A and 8% solvent B was maintained for 2 min. Between 2 and
9 min the percentage of mobile phase B was increased to 35% and then back
to initial the mobile phase composition within 0.1 min, with a total time
of 14 min.

[0128] The column was set at a flow rate of 0.2 ml min-1 and a temperature
of 36° C. Sample volume of 15 μl was used for all LC-MS
experiments. The mass spectrometer was operated in positive electrospray
mode. The capillars temperature was 275° C. and the spray voltage
4.5 kV was used.

[0131] For treatments, cells were seeded at a density of 1×106
cells/3 ml of culture medium in 6-well microtiter plates. Two sets of
experiments were run in parallel for both Confocal Laser Scanning
Microscopy (CLSM) and FACS analysis. For CLSM analysis, on the bottom of
the culture plates there were placed glass coverslips on which the cells
were let grow.

[0132] After 24 hours, the culture medium was changed to media containing
2.5 μM 5-Aza-2-deoxycytidine (5-Aza-2-dC) (Sigma-Aldrich) alone or
3×106 erythro-magneto-HA virosomes containing 5-Aza-dC or
buffer in which the erythro-HA-virosomes were resuspended (control-2).
After 1, 6, 24 and 96 h of incubation, treated cells (with 5-Aza-dC alone
or with erythro-magneto-HA virosomes containing 5-Aza-2-dC or with buffer
in which the erythro-HA-virosomes where resuspended and untreated
(control-1) cells were analyzed by CLSM and FACS analysis.

[0134] Tumor Cells grown on slides in presence of
erythro-magneto-virosomes containing 5-Aza-2-dC were washed in PBS buffer
and fixed with 4% paraformaldehyde in 1×PBS. Cell nuclei were
counterstained with DAPI and mounted in antifade medium. Fluorescent and
brightfield images were obtained by a Leica TCS SP5 inverted microscope
system, equipped with five lasers emitting from the UV to the visible
(405 Diode, Argon, HeNe 543, HeNe 594 and HeNe 633). The DAPI
fluorescence was detected at excitation of 405 nm and emission of 454 nm
while the green fluorescence of superparamangetic nanoparticles at
excitation of 543 nm and emission of 613 nm (see FIGS. 6 and 7).

[0135] Cytofluorimetric Analysis (FACS Analysis)

[0136] FACS analysis was carried out on HeLa cells treated with 2.5 μM
5-Aza-dC or 3×106 erythro-magneto-HA-virosomes containing
5-Aza-2-dC and compared to those untreated (control-1) after 48 and 96
hours of culture. FACS analysis was also carried out on HeLa cells
treated with buffer in which the erythro-HA-virosomes were resuspended to
check the absence of unincorporated 5-Aza-2-dC drug (control-2). Cells
were fixed in ethyl alcohol and the nuclei were stained with 25 mg/ml of
propidium iodide and incubated with 1 mg/ml of RNases for 1 h at
37° C. The nuclear DNA content, which discriminates the cell cycle
phases, was determined using flow cytometry using Becton-Dickinson
FACScan Centroll (see FIG. 8a,b,c).

[0139] To evaluate the in vivo efficiency and efficacy of the present
invention drug delivery system encapsulating therapeutic compounds and to
set the best therapeutic conditions for an innovative therapies based on
erythro-magneto-virosomes delivery system, CD1 nu/nu (nude) mice are
xenografted with human tumor cells and treated with 5-Aza-dC or other
anti-blastic drugs delivered by erythro-magneto-virosomes. To determine
the effect of treatments, the mean tumor mass of treated mice is
evaluated measuring their size with a caliper and by ultrasound.
Additionally, immuno-histochemical analysis is performed on collected
frozen tumor tissues of treated and control mice to evaluate the effects
of treatments at histological level. Moreover, the gene expression
profile on collected frozen tumor tissues of treated and control mice by
using RT-qPCR analysis is analyzed to evaluate the effect of treatment at
the molecular level.

[0141] Tumor Induction in a CD1 Nu/Nu Animal Model and their Treatments.

[0142] CD1 nu/nu (nude) female mice of 6-7 weeks of age, are injected on
their flank with 106 human tumor cells resuspended in Matrigel. The
growth of the tumor is evaluated every three days once the mass is
palpable. These mice are treated with erythro-virosomes delivering
magneto-nanoparticles and therapeutic compounds injected on the tail
following the experimental setting shown in Table 1.

[0144] The 5-Aza-dC treatment is administered at a dose of 2.5 mg/Kg
(around 10 μg/injection) (group 2) by intraperitoneal injection once a
week for a total of four weeks (Alleman et al., 2004). This dose is well
below the limit of toxicity of the drug tolerated in a murine model
(Momparler and Frith, 1981). Erythro-magneto-virosomes and
erythro-magneto-virosomes encapsulating 5Aza-dC are administered by tail
injection and concentrated at tumor mass by applying static magnetic
field (Groups #3 and 4). 1×105 erythro-magneto-virosomes
(group 3) and 1×105 erythro-magneto-virosomes containing
5-aza-dC (group 4) are tail injected with a total amount of 10
μg/injection.

[0145] Number of Mice Involved in the Study

[0146] Four groups of nude mice are involved in the study. One group of
mice is used as a control and receives only the injection of saline
solution (CTRL 1). This control group (Group #1) includes 12 mice each,
divided into other 2 subgroups, which are sacrificed at four and six
weeks following injections of control saline solution. The remaining
three experimental groups (Groups #2, 3 and 4) constituted of 12 mice
each are treated with therapeutic compound such as 5-Aza-dC alone, with
erythro-magneto-virosomes alone and erythro-magneto virosomes
encapsulating therapeutic compound such as 5-Aza-dC (see table 1). Also
these experimental groups are divided into other 2 subgroups, which are
sacrificed at four and six weeks following treatments. The group #2 of
mice receives intraperitonal injection of therapeutic compound as 5-AzadC
at time 0. The group #3 of mice receives tail injection of
erythro-magneto-virosomes at time 0. The group #4 of mice receive tail
injection of erythro-magneto-virosomes encapsulating therapeutic compound
as 5-Aza-dC at time 0.

[0147] These experimental groups include 12 mice per group, each divided
into other 2 subgroups, that are sacrificed at four and 6 weeks following
treatment (6 mice per subgroup×2 sacrification times=total 12 mice
per group; 4 experimental groups×12 mice per group=48 total mice
for each set of experiment). Mice in each subgroup (constituted of 6
mice) are further divided in two other subgroups of 3 mice each (3 mice
for fresh sample collection and 3 mice for paraffin embedded sample
collection) in order to ensure there is enough material to perform the
experiments. The experiments are repeated in the same exact condition if
the first experiment is informative. Animals are housed in micro-isolator
cages within climate-controlled laboratories. All mice are maintained
under identical conditions of temperature (21±1° C.), humidity
(60±5%) and light/dark cycle, and have free access to normal mice
diet. The cages, food and water are inspected on a daily basis and
replenished or changed on needed basis. Animals are monitored daily for
signs of distress and pain by a certified veterinarian. For experiments
involving administration of drugs, animals are anesthetized using
isofluorane. Animals are sacrificed by CO2 asphyxiation as
recommended by the institutional guidelines. Animal weight is monitored
every two days. Tumor volumes are calculated using the formula tumor
volume=(length)×(width)2/2. Alternatively, tumor size is
calculated using a SonoSite ultrasound scanner. Tumor sizes of treated
mice are compared to those of control mice. The tumors are then be
excised and weighed before processing. Tissues that are used for
molecular biological analysis are snap frozen in liquid nitrogen and
stored at -80° C. Tissues to be sectioned are placed in OTC
(Sakura Finetek USA, Inc., Torrance, Calif.), frozen in liquid nitrogen
and stored at -80° C. or preserved in neutral-buffered formalin at
4° C. before embedding in paraffin. The CD1 nu/nu mice are a
suitable host for these studies. The use of animals is necessary because
in vitro techniques cannot fully simulate the interactions between tumor
cells and the tissue environment that plays a pivotal role in tumor
growth. Experimental procedure and justification of the use of the mice
are detailed in table 1.

[0148] All animal experiments in this project are performed at the
Experimental Animal Core facility. The Experimental Animal Core houses
its animals in micro-isolators in the Experimental Animal Facility
adjacent to the research laboratories. An aseptic surgical area adjacent
to the housing room is available for all procedures. The Animal Facility
complies with all necessary national and Institutional animal care and
Biosafety regulations.

[0149] Tissue Collection

[0150] The mice are sacrificed at 4 and 6 weeks post-treatment. Mice are
divided in two groups for sample collection. The samples of the first
group are perfused with 1×PBS, and fixed with 10% formaldehyde.
Tumors are then paraffin-embedded, cut into 5 μm sections and stained
with H&E. The tumor samples of the second group are collected, frozen in
dry ice and kept at -80° C. until use or are placed in OTC (Sakura
Finetek USA, Inc., Torrance, Calif.), frozen in liquid nitrogen and
stored at -80° C. Frozen tumor tissues are either used for western
blot analysis and for RT-qPCR analysis. Tumor size, grade and stage, DNA
ploidy and vascularization, that it is index of the progression of the
tumor, are evaluated (Folberg et al., 1992). Statistic analysis are
conducted through the use of Kaplan-Meier models. The coefficients of
correlation will be calculated using the Spearman coefficient.

[0151] Efficiency and Efficacy of Treatments

[0152] Immuno-Histochemical Analysis.

[0153] Histological sections of the tumor mass are processed for
immunohistochemistry following standard procedures. Formalin-fixed and
paraffin-embedded samples are processed. Sections are mounted on glass
and dried overnight at 37° C. All sections are de-waxed,
rehydrated, quenched in 0.5% hydrogen peroxide and microwave pre-treated
in 10 mM citrate buffer, pH 6.0. After blocking with normal serum for 1
hour at room temperature, tissue sections are incubated with polyclonal
and monoclonal antibodies against tumor markers, either at room
temperatures or at 4° C. for the appropriate time. Negative
controls are produced substituting the primary antibody with pre-immune
serum. All slides are processed by the ABC method (Vector Laboratories,
Burlingame, Calif.). Diaminobenzidine (DAB) is used as the final
chromogen, and Gill's hematoxylin is used as a counterstaining

[0154] Electron Microscopy Analysis

[0155] To evaluate the effective penetration of
"erythro-magneto-virosomes" at cellular level, the tumor tissues from
sacrificed animals are also fixed in 2.5% glutaraldehyde in 0.1 M
cacodilate buffer for 2 hours at 4° C., post-fixed in 1% osmium in
Veronal buffer, dehydrated and embedded in epoxidic resin. The sections
are examined by a Zeiss EM 109 electron microscope.

[0156] RNA Preparation for RT-qPCR Analysis

[0157] Total RNA samples are isolated from tumor masses dissected from the
frozen tumor tissues using TRIZOL reagent (Invitrogen) according to the
manufacturer's instructions. Concentration of purified RNA samples are
determined by A260 measurement and the quality is checked by
Lab-on-a-chip analysis (total RNA nano biosizing assay, Agilent) with the
Agilent 2100 Bioanalyzer.

[0158] Quantitative Real-Time Reverse Transcription-PCR.

[0159] To validate transcriptional responses in the series of treated
animals, tumor biomarkers are evaluated by relatively RT-qPCR following
standard procedures.

[0160] Results

[0161] From the above experimental data it can be outlined that the viral
spike glycoprotein (HA see FIG. 2) purified from influenza virons (FIG.
1) and reconstituted in erythro-magneto particles maintains its fusogenic
properties with the target membrane as shown by the fusion assays in FIG.
4. The yield of therapeutic compound (exemplified by 5-Aza) reconstituted
into erythro-magneto-virosomes is shown in FIG. 5. The total amount of
5-Aza incapsulated in 1106 erythro-magneto virosomes correspond to
0.5 μM. The uptake to the target cell and delivery of the targeted
therapeutic compound is reported in FIGS. 6 and 7. The efficacy of the
active drug (5-Aza) reconstituted in erythro-magneto virosomes to drive
cell towards apoptosis is shown in the cell microscopy (FIGS. 6 and 7)
and in the FACS analysis (see FIGS. 8 a, b and c).

[0162] The results of the experiments indicate that the erythro-magneto
virosomes drug delivery system increases the efficiency and efficacy of
the therapeutic compound treatment. In fact, the total amount of the
therapeutic compound necessary to obtain growth arrest and apoptotic
response in tumor cells is six times less when erythro-magneto virosome
delivery system is used when compared to standard therapy. Moreover, the
anti-neoplastic effect of the exemplified therapeutic compound driven by
the delivery system of the present invention appears 48 hrs earlier than
when standard treatment is applied.